The research described in this proposal is directed towards elucidating the molecular mechanism by which nascent polypeptides are translocated across and integrated into the rough endoplasmic reticulum membrane. Particular emphasis will be placed upon (a) analyzing structural domains in the Sec61 complex that are required for cotranslational gating of the translocation channel by a ribosome-nascent chain (RNC) complex, (b) obtaining additional structures of yeast protein translocation channels by cryoelectron microscopy, and (c) analyzing the role of the Sec62/Sec63 complex in the integration of multi- spanning membrane proteins. The structure of the Methanococcus jannaschii SecYE2 complex provides an excellent model for the closed conformation of a eukaryotic protein translocation channel. Amino acid residues in Sec61p that are important for the cotranslational translocation pathway will be identified by structure-guided mutagenesis of RNC-contact sites and cytoplasmically exposed Sec61 segments. The role of contact sites in RNC-mediated channel gating will be evaluated using a combination of cell biological and biochemical assays. Insertion of a nascent polypeptide into the signal sequence-binding site of the Sec61 complex is thought to require partial separation of the lateral gate of the translocation channel. The effect of Sec61 lateral gate mutations on the in vivo kinetics and fidelity of translocation channel gating will be analyzed. Cotranslational and posttranslational protein translocation channels will be isolated from wild type and mutant cells for cryoelectron microscopy. RNC-channel complexes will be prepared to obtain mid-resolution structures of translocation intermediates. Although the Sec62/Sec63 complex has a well-described role as a signal sequence receptor for yeast proteins that are translocated by a posttranslational pathway, there is a growing body of evidence that suggests that the Sec62/Sec63 complex may enhance the fidelity of membrane protein integration. Yeast strains that have mutations in the subunits of the Sec62/Sec63 complex will be assayed for the ability to integrate single-spanning and multi-spanning membrane proteins. These studies may provide insight into the in vivo role of the Sec62/Sec63 complex in mammalian organisms. The three research objectives outlined above address poorly understood aspects of the protein translocation reaction. The accurate and efficient biosynthesis of integral membrane proteins, secreted proteins and lysosomal proteins is an essential function in human cells, as well as in simple model organisms like budding yeast. Defects in the modification or folding of proteins in the rough endoplasmic reticulum are responsible for a growing list of human diseases that are termed "ER-quality control" diseases. PUBLIC HEALTH RELEVANCE: The biosynthesis of secretory proteins, lysosomal proteins and integral membrane proteins is of fundamental importance for human health. Recent genetic evidence shows that mutations in the human Sec63 gene can cause autosomal dominant polycystic kidney disease, while overexpression of human Sec62 may be linked to prostate cancer. Defects in the biosynthesis, modification and folding of proteins in the rough endoplasmic reticulum is responsible for a growing number of pathologies that are collectively referred to as "ER quality control" diseases. Our studies, which are directed towards understanding the molecular mechanism of protein translocation and membrane protein integration, will provide insight into these crucial events in protein biosynthesis.